The present invention is related to the analysis and study of proteins and cells. More specifically, the present invention is related to the analysis and study of the effects of proteins and other biological and chemical moieties on cells in wells of a plate where the environment of the cells is manipulated and techniques facilitate the identification and tracking of the individual cells and population of cells in the wells.
The analysis of the effects of proteins and other biological and chemical moieties on cells is an important field for the development of improved therapeutics for human health. The analysis of human and other types of cells requires sophisticated analysis methods. This patent application is directed at the improvement of the protein and cellular analysis methods used fro drug discovery and gene and protein function determination.
Establishing protein function is a key part of any drug discovery effort. Whether performing target discovery, prioritization or validation, screening for novel protein therapeutics, lead optimization, or discerning mechanism of action, the demonstration of functional relevance is essential. The human genome comprises approximately 35,000 genes. These genes produce approximately 300,000 to 500,000 proteins in the human body. The sequence of genes that make up the human genome is known, but only about 20% to 30% of this genome has functional definitions of what the specific gene and its protein products produce in health and disease. In the history of the pharmaceutical industry, drugs have acted upon only 500 protein targets in the human body. Over the next few years, the industry will have 100 to 200 times more protein targets produced through genomics and proteomics efforts. A significant opportunity exists to identify, prioritize, and validate these new protein opportunities through discovering their protein function. This function knowledge can be patented.
Most genomic and proteomic efforts infer disease-linked function by methods based upon homology such as proteinxe2x80x94protein association (e.g. two hybrid systems or mass spectroscopy) or disease-linked expression profiling (e.g. 2D gel electrophoresis or various nucleic acid or protein chip technologies). The challenge of demonstrable functional relevance remains an inevitable downstream step in the development of promising candidates from approaches where function is only inferred. The return on any investment in discovery efforts can be lost when a particularly promising candidate has a weak functional role in a disease-relevant system. Despite these drawbacks, inference-based proteomics has been the mode of choice because of the relatively low throughput of various functional determination systems based upon animal models and gene knock-out technologies.
The technology described herein maximizes the efficiency of proteomic drug discovery by establishing protein function. By screening at the level of protein function itself, loss of time and investment due to subsequent failure in a functional assay is minimized. Putative protein targets are discovered, prioritized and pre-validated at the screening step. A key strength of the technology are biochemical and biological assays with the capacity for multiple assay endpoints with a high degree of quantitative sophistication.
Since the assay created by the technology are nondestructive, a single assay well yields kinetic information about protein-related behavior of a given set of cells over assay periods of seconds to weeks. Subtle changes in cellular response to proteins over time are detected and quantified at resolutions as low as five seconds. The analyses are performed within the same well, on the same set of cells at a single cell resolution. Other endpoints include apoptosis, proliferation, changes in cell morphology, cellxe2x80x94cell interactions, protein expression, and other phenotypical changes as listed in Table 1.
The capacity for measuring protein effects upon co-cultures of multiple types of biologically relevant cell type combinations is created. The number and combination of different cell types that can be assayed is determined by the ability of the image analysis software to distinguish each cell type. Delimiters include cell morphology, motility, fluorescent protein expression, antigen elaboration, as well as assay prehistory.
Sophisticated image analysis algorithms make the quantitation of individual cell behavior possible. This means that each cell in an assay is being observed individually for its response to an exogenously added or transfected protein or protein combination. Quantitation at single cell resolution eliminates the masking of subpopulation effects. This unparalleled quantitative resolution, combined with the capacity for extended kinetic assays, ensure that subtle or short-lived cellular responses to proteins are not overlooked.
The present invention pertains to a method for analyzing the effects of proteins and other biological and chemical moieties in cell (s). The method comprises the steps of placing the cell in a solution that suppresses non-biological movement of the cell. There is the step of using time-lapse imaging to analyze motility of the adherent and non-adherent cell(s) in the solution.
The present invention pertains to a method for analyzing the effects of proteins and other biological and chemical moieties in cell(s). The method comprises the steps of placing cells in a solution having a methyl cellulose concentration on a plate having between 6 and 1,536 wells with corresponding volumes of approximately 1 to 4% of the total volume of the solution. There is the step of imaging the cells over time.
The present invention pertains to an apparatus for aligning light in a well of a plate for holding cells. The apparatus comprises a top portion that is adapted to hold to the plate. The apparatus comprises a bottom portion connected to the top portion that is adapted to extend into the well below a meniscus in the well. The top and bottom portions are made of a transparent material that lets light pass through it and be distributed evenly throughout the bottom of the well.
The present invention pertains to a method for lighting a well. The method comprises the steps of placing a first portion of a light alignment apparatus onto a plate having wells so a second portion of the light alignment apparatus extends into a well and below a meniscus in the well. There is the step of directing light onto the first portion that is evenly distributed by the second portion to the bottom of the well.
The present invention pertains to a method for determining a condition of a cell, both in a normal state and in a state where the cell has been effected by the addition of a protein or other biological or chemical moiety. The method comprises the steps of placing a bead coated with a first material in a well of a plate. There is the step of identifying a second material released by a cell in the well by the second material reacting with the first material.
The present invention pertains to an apparatus for indicating a condition of a cell. The apparatus comprises a bead. The apparatus comprises a layer of a first material that reacts with a second material that is released from the cell when it is the condition, the layer coating the bead.
The present invention pertains to a method for establishing a focus profile of a plate having wells for holding cells. The method comprises the steps of taking images at focal points above a current setting and below the current setting. There is the step of applying an image processing sequence to arrive at a focus for the plate.
The present invention pertains to a method for manipulating cells. The method comprises the steps of guiding a pipette with a controller to a predefined position in a well of a plate having a predetermined cell. There is the step of aspirating the cell with the pipette from the well. There is the step of placing the cell at another location with the pipette at the guidance of the controller.
The present invention pertains to an apparatus for manipulating cells. The apparatus comprises a plate having wells that hold cells. The apparatus comprises a controller. The apparatus comprises a pipette means for aspirating a well and controlled by the controller which guides the pipette means to a predetermined position in a well of the plate having a predetermined cell to aspirate the cell with the pipette from the well and place it at another location.
The present invention pertains to an apparatus for monitoring the effects of proteins and other biological and chemical moieties on cells. The apparatus comprises a plate having wells in which cells are disposed. The apparatus comprises means for causing each cell to move to a corner of each well. The causing means is connected to the plate.
The present invention pertains to a method for monitoring the effects of proteins and other biological and chemical moieties on a cell. The method comprises the steps of loading one cell in each well of a plurality of wells of a plate. There is the step of causing each cell to move to a corner of each well.